1. The Field of the Invention
The present invention relates to a system and method for studying a biological agent. More particularly, the present invention relates to a system and method that uses gene silencing for determining a cellular response to the biological agent.
2. The Related Technology
Recently, a natural cellular regulatory pathway was discovered that uses transcribed microRNA (“miRNA”) in order to control protein production. The miRNA includes a duplex region of sense and antisense RNA. This regulatory pathway uses miRNA in order to target complementary mRNA to inhibit production of the encoded protein. Accordingly, a complex series of proteins are involved in this RNA interfering pathway to inhibit or stop production of the proteins encoded by the mRNA. As such, the process is referred to as RNA interference or RNAi.
Additionally, it has been found that the RNAi pathway can be used with synthetic dsRNA (e.g., siRNA) for silencing genes and inhibiting protein expression. This can allow for siRNA having specific sequences to be produced to target complementary DNA and/or mRNA encoding a specific protein. The siRNA can interact with the natural RNAi pathway to silence a target gene and inhibit production of the encoded polypeptide. The ability to silence a specific gene and inhibit production of the encoded protein has been used for basic research of gene function and cellular pathway analysis.
In order to induce gene silencing, the siRNA needs to be introduced into a cell. While the most common procedures for introducing nucleic acids into cells has been forward transfection, reverse transfection (“RTF”) has been developed more recently and used as an alternative to forward transfection procedures. In certain versions of RTF protocols, a complex of lipid-nucleic acid (e.g., lipoplex) can be prepared and introduced into the test wells of a well plate. Cells are introduced into the test wells with the lipid-nucleic acid complexes, and incubated so that the siRNA can enter the cells. Examples of some RTF protocols can be found in U.S. Pat. No. 5,811,274 to Palsson, U.S. Pat. No. 5,804,431 to Palsson and U.S. Pat. No. 6,544,790 to Sabatini and in U.S. Published Applications 2002/0006664 to Sabatini and 2003/070642 to Caldwell et al. As described in these references, RTF procedures for nucleic acids generally can have fewer steps compared to traditional forward transfection and may offer benefits in attempting to isolate the transfected cells to particular regions of a single surface, such as a glass slide. However, RTF procedures for siRNA have not been optimized to the point of practical application, and improvements in gene silencing efficacy are still needed, especially for situations in which one is experimenting with multiple different siRNAs, different gene targets or different cell lines.
Cellular pathways have also been studied with regard to interactions with biological agents. As such, screening techniques have been used to study the interaction between biological agents and cells. These screening techniques can be used to identify which biological agents, such as drugs, have an effect on a particular cell, or interact with a polypeptide or protein produced by the cell. Additionally, screening techniques can be used with infectious biological agents to identify cell types that may or may not be susceptible to an infection, as well as identify drugs that can treat or prevent such infections. Thus, screening techniques can provide a wide array of valuable information that can be used for treating and/or preventing diseases. However, new screening techniques for studying biological agents continue to be developed in order to test various physiological responses.
Therefore, it would be advantageous to have an RTF protocol that can be used to determine the effect of biological agents on cells in the presence of gene silencing. Additionally, it would be beneficial to develop a method to use gene silencing in order to identify genes that encode for gene products that interact with biological agents, such as physiologically active agents or infectious agents.